Anaplasma phagocytophilum (Aph) is a tick-transmitted obligate intracellular bacterium of the family Anaplasmataceae that can infect humans, livestock, companion animals, and wild animals. In addition to Aph, the Anaplasmataceae family members include Anaplasma marginale, Anaplasma platys, Ehrlichia chaffeensis, Ehrlichia canis, and Ehrlichia ruminatium, among others, and all of these cause similar infections known collectively as ehrlichiosis or anaplasmosis. When humans contract an Aph infection, it is more specifically known as human granulocytic anaplasmosis (HGA) and is marked by fever and increased susceptibility to potentially fatal opportunistic infections. Other possible disease manifestations include leukopenia, thrombocytopenia, and elevated serum transaminase levels.
Unfortunately, no vaccines against these infectious agents are currently available. Instead, antibiotic treatments with doxycycline or tetracycline are currently the frontline treatments for anaplasmosis/ehrlichiosis and can be effective. However, the overuse of antibiotics is currently of great concern, and many individuals, such as those that are allergic to the drug, pregnant women, and small children, either cannot or are not advised to take doxcycline. Also troubling is the lack of tools for rapid and definitive diagnosis of anaplasmosis/ehrlichiosis in any species other than dogs.
HGA is an emerging and potentially fatal disease transmitted by the same vectors that transmit Lyme Disease, primarily ticks and deer, but other animal and human hosts can complete the vector cycle and therefore extend the spread of disease. In the U.S., HGA occurs primarily in the Northeast, Upper Midwest, and Northern California. Since HGA became a reportable disease in the U.S. in 1999, the number of cases has risen annually, reaching 2,037 in 2013. Since diagnostic tools for HGA are lacking, the number of actual cases is likely to be much higher. HGA is increasingly recognized in Europe and Asia, and Aph infection is now the most widespread tick-transmitted disease of animals in Europe. Domestic animals, such as dogs and cats, and livestock, such as sheep can be become infected.
As the name implies, an obligate intracellular bacterium must enter a target cell in its human or animal host to survive, replicate, and move to the next host. When an Anaplasma spp. or Ehrlichia spp. infected tick bites a subject, the bacteria is transferred from the tick salivary glands into the tissues of the host or into the bloodstream where they bind to the surface of host cells (especially neutrophils) and are internalized by being taken up into vacuoles that form around each bacterium. For A. phagocytophilum, optimal invasion of mammalian host cells is known to involve multiple cell surface proteins (various adhesins and invasins) which function cooperatively. Within the cell, a resident bacterium prevents the vacuole from merging with lysosomes, which would otherwise destroy the bacterium. In doing so, the bacterium converts the cell into a protective niche that favors bacterial survival and completion of its zoonotic cycle. For example, A. phagocytophilum undergoes a biphasic developmental cycle within neutrophils in which it transitions between an infectious morphotype (dense-cored cell; DC) and a non-infectious, replicative morphotype (reticulate cell; RC). Subsequently, if the Aph infected host is bitten by a tick, DC cells from the host can be ingested by the tick during the blood meal, and the disease is then transferred to new hosts when the infected tick takes another blood meal. However, HGA can also be transmitted perinatally and by blood transfusion, and possibly nosocomially.
While the hallmark of Aph infection is Aph colonization of neutrophils, Aph has also been detected in the microvascular endothelium of heart and liver in experimentally infected severe combined immunodeficiency mice. For experimental purposes, promyelocytic and endothelial cell lines are also useful as in vitro models for studying Aph-host cell interactions. It has been shown that when naïve neutrophils or HL-60 cells are overlaid on Aph-infected endothelial cells, the bacterium rapidly transmigrates into the myeloid cells, demonstrating that Aph infects endothelial cells in vitro and suggesting that the bacterium may transmigrate between endothelial cells and neutrophils during the course of mammalian in vivo infections.
As noted above, humans are not the only species that are infected by members of the Anaplasmataceae family. For example, A. marginale infects bovine erythrocytes. Bovine anaplasmosis causes severe economic loss in many countries, mainly due to the high morbidity and mortality in susceptible cattle herds. Several parameters contribute to the losses due to anaplasmosis and include: low weight gain, reduction in milk production, abortion, the cost of anaplasmosis treatments, and mortality. The losses incurred by the U.S. cattle industry as a result of anaplasmosis are estimated to be $300 million per year whereas in Latin America, losses are calculated to be approximately $800 million per year.
Proteins which mediate the infection of endothelial cells and/or the dissemination of bacteria into the microvasculature of heart and liver are attractive targets for preventing disease caused by Anaplasmataceae bacteria such as Aph. In particular, targeting Aph proteins that are conserved among related Anaplasmataceae family members may also reduce or block transmission of disease caused by a broad spectrum of Anaplasmataceae species. In addition, it would be advantageous to have available a rapid and highly accurate diagnostic test for detecting Anaplasmataceae species infection, especially one that allowed detection early in infection. Unfortunately, no FDA-approved vaccines against Anaplasmataceae species having purified antigens are currently available.
The current gold standard serologic test for diagnosis of anaplasmosis in humans is indirect immunofluorescence assays (IFA) performed as timed pairs over a period of a few weeks, only available in specialized reference laboratories. This assay measures non-specific increases in IgM and IgG antibody levels. However, IgM antibodies, which usually rise at the same time as IgG near the end of the first week of illness and remain elevated for months or longer, are even less specific than IgG antibodies and more likely to result in a false positive. Serologic tests based on enzyme immunoassay (EIA) technology are available from some commercial laboratories. However, EIA tests are qualitative rather than quantitative, meaning they only provide a positive/negative result, and are less useful to measure changes in antibody titers between paired specimens. Furthermore, some EIA assays rely on the evaluation of IgM antibody alone, which again may have a higher frequency of false positive results. Between 5-10% of currently healthy people in some areas may have elevated antibody titers due to past exposure to Aph or to other Anaplasmataceae family members. If only one sample is tested, it can be difficult to interpret. A four-fold rise in antibody titer is needed to achieve significance in paired samples taken weeks apart. Thus, tools for a rapid and definitive diagnosis in any species other than dogs are lacking.
U.S. Pat. No. 7,906,296 B2 to Beall et al teaches polynucleotide sequences from major outer surface protein P44 of Anaplasma platys (Apl), which causes tick-born anaplasmosis in dogs. P44 and peptides from the translated protein can be used for detection of Apl and Aph infection and/or to elicit an immune response in vivo and confer resistance to anaplasmosis caused by Apl or Aph.
U.S. Pat. Nos. 8,158,370; 8,303,959 and US 2013/0064842, all to Liu et al, teach the use of P44 surface proteins from various strain variants of Aph to diagnose and protect against anaplasmosis.
U.S. Pat. No. 8,609,350 B2 to Liu et al. teaches polypeptides from Aph to diagnose and protect against anaplasmosis. The Aph sequences were derived from APH_0915, which encodes a hypothetical open reading frame of a protein of unknown function.
PCT/US2013/047325 to Carlyon, which is herein incorporated by reference, relates to the invasin proteins OmpA and Asp14, and fragments thereof comprising conserved invasin domains. The proteins and fragments are used as therapeutic and diagnostic agents for A. phagocytophilum infection.
Nelson, C. M., et al. (BMC Genomics 9, 364; 2008) used a whole genome transcriptional profiling tiling array analysis to detect A. phagocytophilum genes that are upregulated in vitro during infection of mammalian versus tick cell lines. Many of the proteins encoded by the identified genes were assigned a designation of “hypothetical protein” and of these, several had a predicted cellular location of “outer membrane”, among them APH0915. However, no further information about this putative protein was provided, and no confirmation of its status as an outer membrane protein or possible significance was shown.
A need remains in the art for immunogenic compositions and vaccines to combat Anaplasmataceae infections and for methods to rapidly and accurately diagnose new cases of these diseases, especially with respect to Anaplasmataceae infections that cause anaplasmosis and HGA.